Production of extraneous proteins using plant cultured cells is proceeding. For example, attempts are being made to produce the following proteins useful for humans using tobacco cultured cell:
GM-CSF (see, E. A. James, C. Wang, Z. Wang, R. Reeves, J. H. Shin, N. S. Magnuson and J. M. Lee, “Production and Characterization of Biologically Active Human GM-CSF Secreted by Genetically Modified Plant Cells”, Protein Expr. Purif., 19, 131-138 (2000)), IL-2 and IL-4 (see, N. S. Magnuson, P. M. Linzmaier, R. Reeves, G. An, K. HayGlass and J. M. Lee, “Secretion of Biologically Active Human Interleukin-2 and Interleukin-4 from Genetically Modified Tobacco Cells in Suspension Culture”, Protein Expr. Purif., 13, 45-52 (1998)), immunoglobulin (see, N. S. Magnuson, P. M. Linzmaier, J. W. Gao, R. Reeves, G. An and J. M. Lee, “Enhanced Recovery of a Secreted Mammalian Protein from Suspension Culture of Genetically Modified Tobacco Cells”, Protein Expr. Purif., 7, 220-228 (1996)), erythropoietin (see, S. Matsumoto, A. Ishii, K. Ikura, M. Ueda and R. Sasaki, “Expression of Human Erythropoietin in Cultured Tobacco Cells”, Biosci. Biotechnol. Biochem., 57, 1249-1252 (1993)), and α1-antitrypsin (see, M. Terashima, Y. Murai, M. Kawamura, S, Nakanishi, T. Stoltz, L. Chen, W. Drohan, R. L. Rodriguez and S. Katoh, “Production of Functional Human α1-Antitrypsin by Plant Cell Culture”, Appl. Microbiol. Biotechnol., 52, 516-523 (1999)).
On other hand, it is reported that plant cultured cells secrete many proteins or glycoproteins (see, A. Sturm, “Heterogeneity of the Complex N-Linked Oligosaccharides at Specific Glycosylation Sites of Two Secreted Carrot Glycoproteins”, Eur. J. Biochem., 199, 169-179 (1991); Y. Okushima, N. Koizumi, T. Kusano and H. Sano, “Secreted Proteins of Tobacco Cultured BY2 Cells: Identification of A New Member of Pathogenesis-Related Proteins”, Plant Mol. Biol., 42, 479-488 (2000); and Y. Okushima, N. Koizumi, T. Kusano and H. Sano, “Glycosylation and Its Adequate Processing is Critical for Protein Secretion in Tobacco BY2 Cells”, J. Plant Physiolo., 154, 623-627 (1999)). Of these, in the case of tobacco BY2 cultured cells, two kinds of peroxidases are purified and their genes have been cloned (see, H. Narita, Y. Asaka, K. Ikura, S. Matsumoto and R. Sasaki, “Isolation, Characterization and Expression of Cationic Peroxidase Isozymes Released into the Medium of Cultured Tobacco Cells”, Eur. J. Biochem., 228, 855-862 (1995)). It is also reported that by adding polyvinylpyrrolidone (PVP) to the medium, the concentration of protein secreted in the medium could be increased (see, N. S. Magnuson, P. M. Linzmaier, J. W. Gao, R. Reeves, G. An and J. M. Lee, “Enhanced Recovery of A Secreted Mammalian Protein from Suspension Culture of Generically Modified Tobacco Cells”, Protein Expr. Purif., 7, 220-228 (1996)) and by Y. Okushima et al., supra. (1999) that from tobacco BY2 strain cultured cells, hundreds of proteins are extracellularly secreted. Among these, extracellular secretion of many glycoproteins are confirmed because of their reaction with lectin (concanavalin A) which recognizes high mannose-type sugar chains (see, Y. Okushima, supra. (1999)).
With respect to these glycoproteins, in particular, immunoglobulin, interleukin and GM-CSF, produced within plant cells, a signal peptide of each glycoprotein itself is also recognized in the secretion mechanism within the plant cell, and is secreted in the culture solution (see, E. A. James et al., supra.; N. S. Magnuson et al., supra. (1998); and N. S. Magnuson et al., supra. (1996)). In any of these glycoproteins, it is suggested, the sugar chain participates in the determination of half-life in blood, sensitivity to protease and stability. However, the sugar chain structures of recombinant proteins actually produced within plant cells and purified have not been examined and these proteins are presumed to have a plant-type sugar chain structure.
In the analysis of sugar chain structure, the secretion-type antibody molecule sIgA, produced from tobacco plants is revealed to have a plant-type sugar chain (see, M. Cabanes-Macheteau, A. C. Fitchette-Laine, C. Loutelier-Bourhis, C. Lange, N. D. Vine, J. K. Ma, P. Lerouge and L. Faye, “N-Glycosylation of a Mouse IgG Expressed in Transgenic Tobacco Plants”, Glycobiology, 9, 365-372 (1999)). Furthermore, in the case where another antibody molecule is produced from the same tobacco plant body, the antibody protein produced within the cell is decomposed by the protease and is unstable (see, L. H. Stevens, G. M. Stoopen, I. J. Elbert, J. W. Molthoff, H. A. Bakker, A. Lommen, D. Bosch and W. Jordi, “Effect of Climate Conditions and Plant Developmental Stage on the Stability of Antibodies Expressed in Transgenic Tobacco”, Plant Physiol., 124, 173-182 (2000)). By the Western method using an antiplant-type sugar chain antibody, addition of a plant-type sugar chain to this antibody is confirmed. Although it is reported that the β1,4-linked galactose residue present in the sugar chain of an antibody molecule produced by human or mouse contributes to the stabilization of antibody protein, this sugar residue is absent in the antibody molecule produced by plant cells. Because of this, the antibodies produced by tobacco plants are considered to be prone to decomposition by the protease.
In the case where erythropoietin is produced by tobacco cultured cells, the biological activity is recognized in vitro, but the activity in vivo is not detected (see, S. Matsumoto, K. Ikura, M. Ueda and R. Sasaki, “Characterization of a Human Glycoprotein (Erythropoietin) Produced in Cultured Tobacco Cells”, Plant Mol. Biol., 27 1163-1172 (1995). This is concluded to occur because erythropoietin of which sugar chain is considered to greatly participate in the biological activity, has a largely different sugar chain structure when produced by plant cells.
On the other hand, it is suggested that the plant-type sugar chain may be an allergen in mammals including humans. That is, the sugar chain structure peculiar to plants, such as β1,2-xylose and α1,3-fucose which are not seen in glycoproteins of mammals, are reported to act as an allergen (see, K. Fotisch, F. Altmann, D. Haustein and S. Vieths, Involvement of Carbohydrate Epitopes in the IgE Response of Celery-Allergic Patients, Int. Arch. Allergy Immunol., 120, 30-42 (1999): I. B. Wilson, J. E. Harthill, N. P. Mullin, D. A. Ashford and F. Altmann, “Core α1,3-Fucose is a Key Part of the Epitope Recognized by Antibodies Reacting Against Plant N-Linked Oligosaccharides and is Present in a wide Variety of Plant Extracts”, Glycobiology, 8, 651-661 (1998); and R. van Ree, M. Cabanes-Macheteau, J. Akkerdaas, J. P. Milazzo, C. Loutelier-Bourhis, C. Rayon, M. Villalba, S. Koppelman, R. Aalberse, R. Rodriguez, L. Faye and P. Lerouge, “β(1,2)-Xylose and α(1,3)-Fucose Residues Have a Strong Contribution in IgE Binding to Plant Glycoallergens”, J. Biol. Chem., 275(15), 11451-11458 (Apr. 14, 2000). Accordingly, proteins for medical uses must have a sugar chain structure free of β1,2-xylose or α1,3-fucose.